1. Field of the Invention
The present invention relates to reagents for assaying lipids containing an esterase, more particularly, to reagents for assaying neutral fats, total cholesterols, high-density lipoprotein cholesterols, and/or low-density lipoprotein cholesterols that can be used in the field of clinical chemistry.
2. Description of the Related Arts
‘Esterase’ is the generic name for enzymes that hydrolyzes esters among hydrolases, is classified in the group EC3.1, and includes lipases and cholesterol esterases. ‘Lipase’ is an enzyme that is also called ‘glycerol ester hydrolase’, hydrolyzes glycerol esters, and releases fatty acids, and includes many lipases having different substrate-specificities and localizations such as pancreatic lipase, lipoprotein lipase (LPL), hepatic triacyl glycerol lipase (HTGL), phospholipase, glycolipid-degrading lipase, sphingolipid-degrading lipase, and hormone-sensitive lipase.
Esterase is widely used for assaying lipid components such as neutral fats, cholesterol, phospholipids, glycolipids, sphingolipids, and lipoproteins in a specimen in the field of clinical test that covers assaying specific component(s) in a biological specimen.
Reagents that permit accurate and precise measurements are required in the field of clinical test. One of factors that disturb the accurate assay is the turbidity of biological specimens. The main reason for the turbidity of biological specimens is often chylomicron and ultra low-density lipoprotein that are lipoproteins. These lipoproteins contain neutral fats that are non-polar lipids at high levels, so that they often give turbidity in an aqueous solution. A method was disclosed in which a lipase is added to the reagent in order to solubilize the lipoprotein and to avoid its influence (Patent reference 1). As methods for avoiding the influence of the turbidity of a specimen, techniques that permit solubilizing, by various surfactants, the lipoprotein that causes the turbidity, have also been disclosed (Patent references 2–4). Since a method in which a lipase is added to a reagent cannot be used for a method for assaying lipoprotein(s) and/or lipid component(s) in which a lipase is used as a reactive component, a method in which a surfactant is added is used.
Another factor that prevents the accurate analysis is the stability of reagents. Many reagents that are used for the clinical test are provided as liquids or a lyophilized state. A lyophilized reagent is dissolved in a predetermined solution before use. After measurement, the remaining reagent is stored at a cool place until the next use. Therefore, the stability of a reagent and a liquid product until delivered for the use and/or during storage, and/or the stability of the solution when used, must be adequately secured. It is well known, however, that enzymes such as esterase are unstable in general. Under the present situation, this problem is dealt with by shortening the expiration date for use of a liquid product or a solution prepared by dissolving a lyophilized preparation, or by adding stabilizer(s). For example, when an esterase is used as a reagent, a surfactant that is expected to have a stabilizing effect may be used. It is believed that an esterase requires an interface (between water and oil) as a place for reacting with a lipid, so that surfactant(s) is/are often added to a reagent containing the esterase in order to create such a place to enhance the enzymatic activity of the lipase.
Patent reference 1Japan Patent Laid-Open Pub. No. Hei 09-288111Patent reference 2Japan Patent Laid-Open Pub. No. Sho 59-162454Patent reference 3Japan Patent Laid-Open Pub. No. Sho 61-95247Patent reference 4Japan Patent Laid-Open Pub. No. 2001-188065